Our ongoing clinical trials of immunotherapy for pediatric sarcomas are targeting the tumor specific fusion proteins generated by the t(11;22) and t(2;13) in Ewing's sarcoma and alveolar rhabdomyosarcoma respectively. Thus far, we have had difficulty generating measurable immune responses toward these targets. In an attempt to potentially identify new targets for immunotherapy and to investigate whether endogenous immune responses might already exist in patients with Ewing's sarcoma, we have conducted a series of experiments to study endogenous T cell mediated immunity which is directed toward autologous tumors are present in patients presenting with Ewings sarcoma. Autologous T cells were collected by apheresis from patients with Ewing's sarcoma and were tested for reactivity toward autologous tumors. These studies showed that cytolytic T cells are circulate in patients with Ewing's sarcoma. These tumor reactive killers can be expanded and activated by CD40L-matured DCs following by re-stimulation with tumor cells, or anti-CD3 and 4-1BB antibodies in the absence of autologous tumor. Using such methods, we generated tumor-lytic T cell responses in 5/5 patients. While the tumor lysis was only MHC restricted in some patients, we also observed tumor induced proliferation which showed consistent MHC restriction. Furthermore, using PCR, we identified activation NK receptors on the lytic T cells, provided a basis for the promiscuous cytotoxicity. Phenotypically, the tumor reactive cell lines are predominantly CD3+CD8+CD28-. Furthermore,CD8+/CD28-/4-1BB (CD137)+ produce IFNg in response to autologous tumor whereas CD8+/CD28-/4-1BB- cells do not. 4-1BB is a member of the tumor necrosis factor receptor (TNFR) superfamily that is preferentially expressed on the surface of CD8+ T cells after antigen-induced activation. Cross-linking of 4-1BB and T cell receptor (TCR) on activated T cells has been shown to deliver a costimulatory signal to T cells. Thus, during the course of these studies we investigated whether Ewing's sarcoma itself might be capable of providing the 4-1BB mediated costimulatory signal. We discovered that Ewing's sarcoma cell lines themselves express 4-1BB ligand. Furthermore, T cell activation could be induced by the tumor alone but required both signal one (presumably provided by a tumor associated antigen) and signal two which is provided by 4-1BB ligand expressed by the tumor. When either of these interactions are blocked, the tumor induced proliferation of autologous T cells is blocked. Finally, using a SCID-hu PBL model wherein autologous Ewings tumors are xenografted onto the extremity of mice, we observed diminished growth of primary tumors and diminished metastatic disease when anti-CD3/4-1BBL tumors we administered compared to anti-CD3/anti-CD28 or compared to administration of non-stimulated CD8+ T cells. These results provide new evidence that endogenous immune responses exist in patients with Ewing's sarcoma and question the widely held view that progressive tumor growth induces T cell tolerance. Furthermore, these results open the intriguing possibility that tumor expression of an immune mediated co-stimulatory molecule may provide ongoing signals which sustain an immune response in vivo. We are currently optimizing approaches to expand the CD3+CD8+4-1BB+ subset from both normal hosts and tumor bearing hosts and can currently accomplish a two log expansion of these cells. These results are currently being submitted for publication [Tumor Expression of 4-1BB Ligand: Sustenance for Tumor Specific Cytolytic T Cells? Hua Zhang, Kevin S Chua, Chand Khanna Lee J Helman, Bill Telford, Yvona Ward, Elaine K Thomas and Crystal L Mackall, submitted.] These studies were carried out in part through a CRADA developed with Immunex Corp. to obtain CD40 ligand for dendritic cell differentiation as well as various reagents for blocking 4-1BB/4-1BBL interactions. We are also currently studying the biology of programmed cell death in Ewings sarcoma in an effort to identify potential blocks in death pathways which may modulate susceptibility to immune based therapies. Previously, our group published evidence for susceptibility to Fas mediated cell in Ewings sarcoma and refuted the possibility that Fas ligand by Ewings sarcoma might impact immune susceptibility. Due to the toxicity of Fas based therapies however, clinical application of Fas ligation is not practical. However, through a collaboration with Udo Kontny, Univ. of Frieburg, Germany, we discovered that Ewings sarcoma is also quite susceptible to programmed cell death via ligation of TRAIL receptors -1 and -2 (Kontny HU, Hammerle K, Shayan P, Mackall CL and Niemeyer CM. Sensitivity of Ewing's sarcoma to TRAIL-induced apoptosis. Cell Death and Differentiation, 2001;8:506-514). Current studies are underway to determine whether this susceptibility in vitro translates into efficacy of TRAIL based therapies in vivo using murine xenografts. These studies have confirmed evidence for activity in vivo and are currently being explored further. During the course of these studies, we also made the surprising observation that Ewings sarcoma is induced to undergo apoptosis during coincubation with the tyrosine kinase inhibitor STI-571. In collaboration with Dr. Carol Thiele's lab, we characterized this effect and have provided evidence to suggest that it occurs via a pathway independent of c-kit and/or PDGF inhibition.